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D A Shub

Publications and source records attributed to D A Shub.

At least 19 recordsLinked to original sources
PubMed2001

Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites.

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.

Amino Acid Sequence↗
PubMed2001

A novel group I intron-encoded endonuclease specific for the anticodon region of tRNA(fMet) genes.

Open reading frames (ORFs) are frequently inserted into group I self-splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self-splicing intron in the tRNA(fMet) gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intron-encoded endonucleases or maturases. Here, using an in vitro transcription-translation system, we show that this intronic ORF encodes a double-strand DNA endonuclease, I-Ssp6803I. I-Ssp6803I cleaves each strand of the intronless tRNA(fMet) gene adjacent to the anticodon triplet leaving 3 bp 3' extensions and has no activity at intron-exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNA(fMet) gene. I-Ssp6803I represents a novel intron-encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.

Amino Acid Sequence↗
PubMed1999

Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: introns in multiple genes, a single gene with three introns, and exon skipping by group I ribozymes.

Analysis of RNA that can be labeled with GTP indicates the existence of group I introns in genes of at least three transcriptional classes in the genome of Staphylococcus aureus bacteriophage Twort. A single ORF of 142 amino acids (Orf142) is interrupted by three self-splicing group I introns, providing the first example of a phage gene with multiple intron insertions. Twort Orf142 is encoded in a message that is abundant 15-20 min after infection and is highly similar to a late gene product (Orf8) of the morphologically related Listeria phage A511. The introns in orf142 are spliced in vivo and contain all the conserved features of primary sequence and secondary structure of group I introns in subgroup IA2, which includes the introns in Escherichia coli phage T4 and the Bacillus phages beta22 and SPO1. Introns I2 and I3 in orf142 are highly similar, and their intron insertion sites are closely spaced. The presence of transcripts with a skipped exon between these introns indicates that they may fold into a single active ribozyme resulting in alternative splicing. Alternatively, the cleaved 5' exon preceding I2 may undergo trans splicing to the 3' exon that follows I3. Regardless of the detailed mechanism, these results demonstrate a new means whereby a single gene can give rise to multiple messenger RNAs.

Amino Acid Sequence↗
PubMed1999

Sporadic distribution of tRNA(Arg)CCU introns among alpha-purple bacteria: evidence for horizontal transmission and transposition of a group I intron.

A group I intron interrupts the tRNA(Arg)CCU gene of the alpha-purple bacterium Agrobacterium tumefaciens (B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173-176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of alpha-purple bacteria. Of 10 newly identified tRNA(Arg)CCU genes, we found only two that contained an intron homologous to that of the Agrobacterium intron. This restricted and scattered distribution of the tRNA(Arg)CCUg intron among alpha-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA(Leu)UAA introns found in strains of the cyanobacterium Microcystis aeruginosa (K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293-298, 1997) and alpha-purple tRNA(Arg)CCU introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA(Leu)UAA introns.

Agrobacterium tumefaciens↗
PubMed1997

Origin and evolution of group I introns in cyanobacterial tRNA genes.

Many tRNA(Leu)UAA genes from plastids contain a group I intron. An intron is also inserted in the same gene at the same position in cyanobacteria, the bacterial progenitors of plastids, suggesting an ancient bacterial origin for this intron. A group I intron has also been found in the tRNA(fMet) gene of some cyanobacteria but not in plastids, suggesting a more recent origin for this intron. In this study, we investigate the phylogenetic distributions of the two introns among cyanobacteria, from the earliest branching to the more derived species. The phylogenetic distribution of the tRNA(Leu)UAA intron follows the clustering of rRNA sequences, being either absent or present in clades of closely related species, with only one exception in the Pseudanabaena group. Our data support the notion that the tRNA(Leu)UAA intron was inherited by cyanobacteria and plastids through a common ancestor. Conversely, the tRNA(fMet) intron has a sporadic distribution, implying that many gains and losses occurred during cyanobacterial evolution. Interestingly, a phylogenetic tree inferred from intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.

Conserved Sequence↗
PubMed1996

Beyond homing: competition between intron endonucleases confers a selective advantage on flanking genetic markers.

The closely related B. subtilis bacteriophages SPO1 and SP82 have similar introns inserted into a conserved domain of their DNA polymerase genes. These introns encode endonucleases with unique properties. Other intron-encoded "homing" endonucleases cleave both strands of intronless DNA; subsequent repair results in unidirectional gene conversion to the intron-containing allele. In contrast, the enzymes described here cleave one strand on both intron-containing and intronless targets at different distances from their common intron insertion site. Most surprisingly, each enzyme prefers DNA of the heterologous phage. The SP82-encoded endonuclease is responsible for exclusion of the SPO1 intron and flanking genetic markers from the progeny of mixed infections, a novel selective advantage imparted by an intron to the genome in which it resides.

Bacillus Phages↗
PubMed1994

An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.

The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.

Amino Acid Sequence↗
PubMed1994

Self-splicing group I intron in cyanobacterial initiator methionine tRNA: evidence for lateral transfer of introns in bacteria.

A group I self-splicing intron has been found in the anticodon loop of tRNA(fMet) genes in three cyanobacterial genera: Dermocarpa, Scytonema and Synechocystis; it is absent in nine others. The Synechocystis intron is also interrupted by an open reading frame (ORF) of 150 codons. Of these three bacteria, only Scytonema also contains the group I intron that has previously been reported in tRNA(Leu) (UAA) genes in both cyanobacteria and chloroplasts. The presence of an ORF in the tRNA(fMet) intron, the sporadic distribution of the intron among cyanobacteria and the lack of correlation between relatedness of the intron sequences and the bacteria in which they reside, are all consistent with recent introduction of this intron by lateral transfer.

Amino Acid Sequence↗
PubMed1994

The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames.

A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1). In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron. All of these introns have group I secondary structures that are extremely similar to one another in primary sequence. Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged. Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1.

Amino Acid Sequence↗
PubMed1994

Intron-containing T4 bacteriophage gene sunY encodes an anaerobic ribonucleotide reductase.

The function of the SunY protein, encoded by an intron-containing gene of bacteriophage T4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the SunY intron. Here we show that anaerobic T4 infections of Escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme. Inactivation of the T4 sunY gene (in this communication renamed nrdD) significantly decreased both the induced activity and the anaerobic production of phage, confirming the role of the T4 NrdD (SunY) protein as a phage-specific anaerobic ribonucleotide reductase. With the identification of the T4 nrdD (sunY) gene product as an anaerobic ribonucleotide reductase, all known bacteriophage introns are found to share the common and as yet unexplained property of residing within genes of DNA metabolism.

Amino Acid Sequence↗
PubMed1994

Bacterial viruses. Bacterial altruism?

Some strains of Escherichia coli harbor genes that trigger cell death upon infrection by bacteriophage T4; these may provide examples of the evolution of altruistic behavior in bacteria.

Bacteriophage T4↗
PubMed1992

Self-splicing introns in tRNA genes of widely divergent bacteria.

The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions. A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts. But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria. Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria. We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp. (beta). The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide. Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.

Base Sequence↗
PubMed1992

Activation of the catalytic core of a group I intron by a remote 3' splice junction.

Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions. Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo. Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction. Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation. A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing. A role for these interactions in mediating putative alternative splicing events is discussed.

Base Sequence↗
PubMed1991

The antiquity of group I introns.

The recent discovery of self-splicing introns in cyanobacteria has given renewed interest to the question of whether introns may have been present in the ancestor of all living things. The properties of introns in genes of bacteria and bacteriophages are discussed in the context of their possible origin and biological function.

Animal Population Groups↗